1. Field of the Invention
The present invention relates generally to the fields of vector biology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors with replacement of fibers for cell-specific targeting with concomitant elimination of endogenous tropism.
2. Description of the Related Art
Approaches to target adenoviral vectors to specific cell types should be based on an understanding of the mechanism of cell entry exploited by the majority of human adenoviruses and on the identification of the components of the adenoviral virion which are involved in the early steps of the virus-cell interaction. Adenoviruses are non-enveloped viruses containing a double stranded DNA genome packaged into an icosahedral capsid. Whereas the most abundant capsid protein, the hexon, performs structural functions and is not involved in the active cell entry process, the other two major protein components of the capsid, the fiber and the penton base, have been shown to play key roles in the early steps of virus-cell interaction. The fiber and penton base together form penton capsomers consisting of five penton base subunits embedded in the virus capsid tightly associated with a homotrimer of fiber proteins protruding from the virion.
Each of the five subunits of the penton base contains a flexible loop structure, which corresponds to a hypervariable domain of the otherwise highly conserved protein. Amino acid sequence analysis of penton base proteins of different adenoviral serotypes showed that each loop consists of two stretches of alpha helices flanking an arginine-glycine-aspartic acid (RGD) tripeptide positioned in the middle of the loop. Cryo-electron micrography (cryo-EM) studies of Ad2 virions revealed that these loops form 22 xc3x85 protrusions on the surface of penton base, thereby facilitating interaction of the RGD motif, localized at the apex of the protrusion with cellular integrins.
The fiber has a well-defined structural organization with each of its three domains, the tail, the shaft, and the knob, performing a number of functions vital for the virus. The short amino terminal tail domain (46 amino acid residues in Ad2 and Ad5 fibers) of the fiber protein is highly conserved among most adenoviral serotypes. In addition to being involved in the association with the penton base protein through an FNPVYD (SEQ ID NO: 15) motif at residues 11-16, which results in anchoring the fiber to the adenoviral capsid, the tail domain also contains near its amino terminus the nuclear localization signal KRxcexR (where indicates a small amino acid residue), which directs the intracellular trafficking of newly synthesized fibers to the cell nucleus, where the assembly of the adenoviral particle takes place.
The central domain of the fiber is the shaft, which extends the carboxy terminal knob domain away from the virion, thereby providing optimal conditions for receptor binding. The shaft is organized as a sequence of pseudorepeats, each 15 amino acids in length, with a characteristic consensus sequence containing hydrophobic residues at highly conserved positions. This sequence, X-Xxcfx86-X-xcfx86-X-xcfx86-G-X-G-xcfx86-X-xcfx86-X-X or X-X-xcfx86-X-xcfx86-X-xcfx86X-X-P-xcfx86-X-xcfx86-X-X, contains hydrophobic amino acids at xe2x80x9cxcfx86xe2x80x9d-positions, with either the eighth and tenth positions being occupied with two glycines or with a proline in the tenth position. The models for the secondary structure corresponding to these repeats describe the shaft as a triple xcex2-spiral in which the xcex2-strands are oriented more along the fiber axis and the hydrophobic residues at the 7th and 13th position are located at greater radius. The trimer is stabilized with extensive intra- and inter-chain hydrogen bonding. Due to its rod-like shape, the shaft domain basically determines the length of the entire molecule, which depends on the number of pseudorepeats contained within the shaft. The fibers of various human adenoviral serotypes contain different number of repeats, resulting in a significant variation in the fiber length: from 160 xc3x85 (Ad3) to 373 xc3x85 (Ad2 and Ad5).
The carboxy terminal knob domain (180-225 amino acid residues) carries out two distinct functions, i.e., initiation of fiber and trimerization and binding of the virus to its primary cellular receptor. X-ray crystallography studies on E. coli-expressed Ad5 fiber knob protein have shown that the trimeric knob is arranged around a three-fold crystallographic symmetry axis and resembles a three bladed propeller when viewed along this axis. Each monomer of the knob is a xcex2-sandwich structure, formed by two antiparallel xcex2-sheets R and V. The surface of the V-sheet, which consists of the strands A, B, C, and J, points towards the virion, while the R-sheet, formed, by strands D, I, H, and G, points outside the virion and towards the surface of the target cell. These findings have been then corroborated with X-ray crystallography data obtained with recombinant Ad2 fiber knob protein.
A number of studies employing recombinant knobs have shown that these proteins are capable of self-trimerization, which does not require any cellular chaperons. The exact trimerization motif within the fiber knob is largely unknown, which makes mutagenesis or modification of this protein quite difficult: indeed, any new mutation or modification of the fiber may affect amino acid(s) involved in the fiber trimerization and may therefore destabilize the entire molecule, thereby rendering it non-functional. The mutant knobs revealed that deletions in the knob sequence, even as short as two amino acid residues, may result in monomeric fibers, which cannot associate with penton base and, therefore, cannot be incorporated into mature adenoviral particles.
The second function performed by the knob is binding to a cellular receptor and, therefore, mediating the very first step of the virus-cell interaction. This receptor-binding ability of the knob has been demonstrated by utilization of recombinant knob proteins as specific inhibitors of adenoviral binding to cells. Based on the xcex2-sandwich structure of the knob, it was originally hypothesized by Xia et al. that the strands constituting the R-sheet form a receptor binding structure. Recently, however, analysis of fiber knob mutants has revealed that segments outside the R-sheet constitute the receptor-binding site. The Ad5 binding site is located at the side of the knob monomer and specifically involves sequences within the AB and DE loops and B, E, and F xcex2-strands. The binding site of Ad37 that binds to a different receptor involves a critical residue in the CD loop at the apex of the trimer.
The two penton proteins, the penton base and fiber, work in a well-orchestrated manner to provide the early steps of the cell infection mechanism developed by adenoviruses. Importantly, each of these early events is mediated by either fiber or penton base; therefore, both proteins play distinct and well defined roles in this process.
The fiber knob provides the initial high-affinity binding of the virus to its cognate cell surface receptor, coxsackievirus and adenovirus receptor (CAR), which does not possess any internalization functions and merely works as a docking site for Ad attachment.
Human adenoviruses (Ad) of serotype 2 and 5 have been extensively used for a variety of gene therapy applications. This is largely due to the ability of these vectors to efficiently deliver therapeutic genes to a wide range of different cell types. However, the promiscuous tropism of adenovirus resulting from the widespread distribution of coxsackie virus and adenovirus receptor (CAR) (1, 2), limits the utility of adenoviral vectors in those clinical contexts where selective delivery of therapeutic transgene to a diseased tissue is required. Uncontrolled transduction of normal tissues with adenoviral vectors expressing potentially toxic gene products may lead to a series of side effects, thereby undermining the efficacy of the therapy. Furthermore, cell targets expressing CAR below certain threshold levels are not susceptible to adenoviral-based therapies due to their inability to support adenoviral infection. Therefore, the dependence of the efficiency of the adenoviral-mediated cell transduction on the levels of CAR expression by the target cell presents a serious challenge for the further development of adenoviral-based gene therapeutics.
In order to overcome this limitation, the concept of genetic targeting of adenoviral vectors to specific cell surface receptors has been proposed. Strategies to retarget adenoviral vectors are based on the currently accepted model of adenoviral infection (3), which postulates that the initial binding of the adenoviral virion to the cell is mediated by the attachment of the globular knob domain of the adenoviral fiber protein to CAR. This is then followed by an internalization step triggered by the interaction of the RGD-containing loop of a second adenoviral capsid protein, the penton base, with cellular integrins. Although recent studies have shown that representatives of different adenoviral serotypes may utilize cell receptors other than CAR, the two-step mechanism of cell entry established for Ad2 and Ad5 appears to be common to the majority of human adenovirus. As the fiber protein is the key mediator of the cell attachment pathway employed by Ad, genetic incorporation of targeting ligands within this viral protein was originally proposed as the strategy to derive targeted, cell type specific adenoviral vectors.
Although the primary amino acid sequences of fiber proteins of various human and animal adenoviruses are highly diverse, the overall structural and functional organization of these proteins demonstrate remarkable degree of similarity. Indeed, all key features of the domains of the fiber proteins described abovexe2x80x94the presence of the nuclear localization signal and the penton base binding site within the fiber tail; the presence of pseudorepeats in the shaft; the propeller-like structure of the knob; and trimeric configuration of the entire fiber moleculexe2x80x94are highly conserved between various adenoviral serotypes. This overall structural and functional similarity has been exploited by a number of investigators, who succeeded in replacing the entire fiber proteins of one adenoviral serotype with those derived from another serotype, or xe2x80x9cshuffledxe2x80x9d individual domains of the fiber molecule utilizing a variety of structural domains pre-existing in nature.
However, it is of paramount importance to note that fiber shuffling does not overcome the limitations associated with the conserved structure of native fibers: as all the adenoviral fibers characterized so far contain the knob domains of similar structure, which carry out the functions of trimerization and receptor binding, it is logical to assume that replacing those knobs with their structurally similar counterparts derived from other adenoviral serotypes would lead to chimeric molecules inheriting all the drawbacks and structural limitations known for the wild type fibers in the context of incorporation of the cell-targeting ligands within these carrier proteins. The same holds true with respect to shuffling of the full size fibers.
In addition, as all wild type adenoviral fibers have affinity to their cognate receptors, it is rather problematic to create recombinant adenoviral vectors targeted to specific cell surface receptors via the fiber shuffling. This maneuver may change the tropism of the vector, but will never result in an adenoviral vector specifically targeted to the cell of interest. Although ablation of native tropism of adenoviral vector via identification and subsequent elimination of specific amino acids of the fiber protein which mediate binding of the virion to its native receptor is generally viewed as the way of derivation of truly targeted adenoviral vectors, it may have limited utility as the mutated sequences may undergo reversion to the wild type during multiple cycles of virus propagation. Due to its restored ability to bind to its native receptor a virion which genome underwent such a reversion immediately achieves selective advantage over the virions which tropism is restricted to one specific receptor. This selective advantage will eventually result in significant contamination of the vector preparation with virions retaining tropism to receptors different from the target one. Therefore the efficiency of the entire targeting maneuver will b e jeopardized.
Furthermore, many human adenoviruses recognize CAR as the primary binding receptor which is expressed by many different cell types. Taken together with the widespread distribution of adenoviral infections in humans, this has led to the belief that chimeric adenoviral virions incorporating fiber proteins originating from different adenoviral serotypes most likely exist in nature when the same cell in a human body gets infected with two adenoviruses belonging to two different Ad serotypes. Therefore, shuffling the fibers is an experimental realization of the viral chimerizm which takes place naturally.
Attempts to generate adenoviral vectors possessing expanded tropism involved incorporation of short peptide ligands into either the carboxy terminal or so-called HI loop of the knob of the Ad fiber protein. Although these studies demonstrated the feasibility of genetic targeting of Ad and showed the potential utility of such vectors in the context of several disease models (7, 8), further progress in this direction has been hampered by the structural conflicts often observed as a result of modification of the fiber structure. Due to the rather complex structure of the fiber knob domain, even minor modifications to this portion of the molecule may destabilize the fiber, thereby rendering it incapable of trimerization and, hence, non-functional. The upper size limit for a targeting ligand to be incorporated into Ad5 fiber is about 30 amino acid residues (5, 9), which dramatically narrows the repertoire of targeting moieties, thereby limiting the choice of potential ligands and, therefore, cell targets. The task of adenoviral targeting is further complicated by the need to ablate the native receptor-binding sites within the fiber of an adenoviral vector to make it truly targeted. As a result of these limitations, only a handful of heterologous peptide ligands (oligo lysine, FLAG, RGD-4C (SEQ ID NO: 14), RGS(His)6 (SEQ ID NO: 16), and HA epitope) have been successfully used in the context of Ad5 fiber modification during last several years.
The prior art remains deficient in the lack of effective means to produce recombinant adenoviral vectors with combination of novel targeting and ablation of native tropism. The present invention fulfills this longstanding need and desire in the art.
The present invention describes the next generation of recombinant, cell-specific adenoviral vectors. More particularly, the instant specification discloses that there are two aspects to consider in the modification of adenoviral tropism: (1) ablation of endogenous tropism; and (2) introduction of novel tropism. To expand the utility of recombinant adenoviruses for gene therapy applications, methods to alter native vector tropism to achieve cell-specific transduction are necessary. To achieve such targeting, the present invention discloses the development of a targeted adenovirus created b y radical replacement of the adenovirus fiber protein. The fiber protein was replaced with a heterologous trimerization motif to maintain trimerization of the knobless fiber and a ligand capable of targeting the virion to a novel receptor was introduced simultaneously. The present invention thus represents a demonstration of the retargeting of a recombinant adenoviral vector via a non-adenoviral cellular receptor.
In one embodiment of the present invention, there is provided a recombinant adenovirus vector lacking endogenous viral tropism but having novel tropism. The adenovirus vector is modified to produce a replacement adenoviral fiber protein so as to modify viral tropism, wherein the replaced fiber gene comprises the amino-terminal portion of the adenoviral fiber gene including the tail domain, the carboxy-terminal portion of the T4 bacteriophage fibritin gene and a ligand. The fiber replacing protein retains the fiber""s capacity to trimerize. Preferably, the ligand can be a physiological ligand, anti-receptor antibodies or cell-specific peptides. The adenoviral vector may further contains a therapeutic gene such as the herpes simplex virus-thymidine kinase gene.
In another embodiment of the present invention, there is provided a method of killing tumor cells in an individual in need of such treatment, comprising the steps of pretreating said individual with an effective amount of the recombinant adenoviral vector disclosed herein and administering ganciclovir to said individual.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.